Food Yellow4 reprotoxicity in relation to localization of DMC1 and apoptosis in rat testes: Roles of royal jelly and cod liver oil.

Food Yellow 4 (FY4) is a lemon-yellow-colored synthetic organic azo dye, which is used widely for imparting pleasant and attractive appearance to foods and cosmetics. The present study aimed at evaluating the possible mechanism underlying the FY4-induced reprotoxicity in rats, and the potential supportive role of royal jelly (RJ) or cod liver oil (CLO), which is a natural remedy with several pharmacological benefits, against induced toxicity. Forty-eight male rats were divided into different groups-the control group, the CLO group (0.4 mL/kg), the RJ group (300 mg/kg), the FY4 group (500 mg/kg b.w.), and the co-treated groups (FY4 + CLO or FY4 + RJ). Semen analysis, serum hormones, and enzyme activities were estimated. Immunohistochemical staining was performed using anti-PCNA, anti-Sox 9, anti-STRA8, anti-DMC1, and anti-ssDNA antibody. The FY4 group exhibited a significant decrease in sperm concentration and motility percentage (%) and a substantial reduction in the TES and LH levels. Testicular LDH, ACP, and SDH were observed to be inhibited. Furthermore, co-localization of DMC1 and ssDNA, which reflected apoptotic induction in the leptotene and zygotene spermatocytes, respectively, was observed to have markedly elevated in the FY4 treated rats, with fewer PCNA-positive and SOX9-positive cells and higher ssDNA-positive cells in the seminiferous epithelium in comparison to the control groups. Interestingly, co-treatment with CLO or RJ exhibited healthy sperms and restored their features, activated the enzyme production, and raised the levels of sexual hormones. In addition, both RJ and CLO restored the features of the testicular tissue as observed under a light microscope, and limited the apoptosis as observed through antibody staining. Collectively, the results of the present study revealed that the co-administration of RJ or CLO with FY4 improved the biochemical, hormonal, and structural aspects of the testicular tissue in rats. Therefore, CLO and RJ may be considered promising agents that would be able to improve the testicular structure and function in the FY4-exposed individuals.

Hibiscus sabdariffa L. as a source of nutrients, bioactive compounds and colouring agents.

The nutritional and bioactive composition of plants have aroused much interest not only among scientists, but also in people's daily lives. Apart from the health benefits, plants are a source of pigments that can be used as natural food colorants. In this work, the nutritional composition of Hibiscus sabdariffa L. was analysed, as well as its bioactive compounds and natural pigments. Glucose (sugar), malic acid (organic acid), α-tocopherol (tocopherol) and linoleic acid (fatty acid) were the major constituents in the corresponding classes. 5-(Hydroxymethyl) furfural was the most abundant non-anthocyanin compound, while delphinidin-3-O-sambubioside was the major anthocyanin both in its hydroethanolic extract and infusion. H. sabdariffa extracts showed antioxidant and antimicrobial activities, highlighting that the hydroethanol extract presents not only lipid peroxidation inhibition capacity, but also bactericidal/fungicidal inhibition ability for all the bacteria and fungi tested. Furthermore, both extracts revealed the absence of toxicity using porcine primary liver cells. The studied plant species was thus not only interesting for nutritional purposes but also for bioactive and colouring applications in food, cosmetic and pharmaceutical industries.

The hazardous effects of three natural food dyes on developmental stages and longevity of Drosophila melanogaster.

Nowadays, food dyes obtained from herbal, animal, microbial and mineral sources are widely used as food additives. In this study, the toxic effects of three different natural food dyes (carmine, turmeric and annatto) on 72 ± 4 h larvae of Oregon-R wild type of Drosophila melanogaster were investigated. For this purpose, four different application doses (50, 75, 100, 125 mg mL(-1)) were chosen by means of preliminary studies. It was determined that larval mortality increased with increasing concentration in the application groups and the toxicity order was carmine > turmeric > annatto. It was observed that the survival rate was highest in the control with 98% and lowest in 125 mg mL(-1) carmine with 16%. In addition, the average lifespan of the adult individuals obtained from third instar larvae was also studied. While the average lifespan was 40.88 ± 1.44 days in the control group, these values were 10.81 ± 0.55-23.90 ± 1.27 days in the carmine group, 15.00 ± 0.80-22.42 ± 1.43 days in the turmeric group and 10.33 ± 1.03-35.68 ± 1.54 days in the annatto group, respectively. According to the obtained results, when both the developmental period from larvae into adults and the lifespan of the developing adults were compared with the control group, the food dyes were found to be toxic and the toxicity order of carmine > turmeric > annatto was identified.

Evaluations of the mutagenicity of a pigment extract from bulb culture of Hippeastrum reticulatum.

The use of anthocyanins in food products as colorants has been limited because of their instability toward alkaline pH and high temperature. This study aimed to determine color stability and mutagenicity of the anthocyanin-based pigment extract from bulb cultures of Hippeastrum (Hippeastrum reticulatum). The pigment extract retained its reddish-orange color under alkaline conditions (⩽pH 11) and was stable up to 6 h at 95 °C. The mutagenicity of the extract was evaluated in vitro and in vivo. Hippeastrum pigment extract up to 1.25 mg plate(-1) was found non-mutagenic in Ames test using Salmonella typhimurium strain TA98 and TA100. Chromosome aberrations were observed when human lymphocytes were treated with the extract up to 1.5 mg ml(-1). However, the extract up to 1.4 mg ml(-1) was found to exhibit relatively low or no mutagenicity in in vitro comet assays with human lymphocytes. In in vivo micronucleated reticulocyte assay, mice were treated orally with the extract up to 1 g kg(-1). No significant increase of the percentage of micronucleated peripheral reticulocytes compared to the negative control groups was found. Taken together, our study indicates that Hippeastrum pigment extract is potentially applicable as an additive colorant in the diet and related products.

The modifying effect of selenium and vitamins A, C, and E on the genotoxicity induced by sunset yellow in male mice.

The use of food additives in various products is growing up. It has attracted the attention towards the possible correlation between the mutagenic potential of food additives and various human diseases. This work evaluated the protective role of selenium and vitamins A, C and E (selenium ACE)(1) against the genotoxic effects induced by a synthetic food additive, sunset yellow, in mice. Six groups were studied including two control groups (negative and positive control), two groups are given single dose of sunset yellow (either 0.325, 0.65 or 1.3mg/kg body weight(2) alone or with selenium ACE) and two groups are given sunset yellow daily for 1, 2 or 3 weeks (0.325mg/kg b.wt./day alone or with selenium ACE), respectively. The study examined the induction of sister chromatid exchanges (SCE's)(3) in bone-marrow cells, chromosomal aberration in somatic (bone-marrow) and germ cells (spermatocytes) after single and repeated oral treatment, and the induction of morphological sperm abnormalities. The results showed that sunset yellow had genotoxic effects as indicated by increased frequency of SCE's, by chromosomal aberrations in both somatic and germ cells, and by increased morphological sperm abnormalities and DNA fragmentation. The results also indicated that the oral administration of selenium ACE significantly reduced the genotoxic effects of sunset yellow, a result that may support the use of antioxidants as chemopreventive agents in many applications.

Contact sensitizing potential of annatto extract and its two primary color components, cis-bixin and norbixin, in female BALB/c mice.

The present studies were performed to examine the contact allergenic effects of an annatto extract (ANT) in female BALB/c mice. ANT at 5-10% induced a greater than threefold increase in lymph node cell proliferation when compared to the control in the LLNA. Moreover, a significant increase in the percent ear swelling at 24h after ANT challenge was observed in the MEST. A significant increase in the percentage of B cells was also observed. To determine which of the two predominant coloring components (norbixin and bixin) in ANT was responsible for the sensitizing effects of ANT, norbixin was subsequently examined, with negative results being observed in both the LLNA and MEST following treatment with norbixin (1-20%). These findings suggested that perhaps bixin was responsible for the positive responses in both the LLNA and MEST following exposure to ANT. Therefore, further studies using a partially purified cis-bixin extract were conducted. Positive responses in both the LLNA and MEST were observed in mice treated with cis-bixin at the concentrations as low as 0.1-0.5%. These results have demonstrated that cis-bixin, but not norbixin, is likely a contact sensitizer and contributes to the contact hypersensitivity effects observed following dermal exposure to ANT in mice.

Safety assessment of dietary administered paprika color in combined chronic toxicity and carcinogenicity studies using F344 rats.

Combined chronic toxicity and carcinogenicity studies of paprika color, used as a food additive in various countries, were performed in male and female F344 rats. Dietary concentrations of 0%, 0.62%, 1.25%, 2.5% and 5% were applied in a 52-week toxicity study and 0%, 2.5% and 5% in a 104-week carcinogenicity study. Treatment with paprika color caused a significant increase in incidence of hepatocellular vacuolation in 5% males, but no toxicological effects were found with reference to survival rates, body weights, hematological or serum biochemical parameters and organ weights at any dose level in either sex in the chronic toxicity study. Also, paprika color did not induce specific tumors nor did it exert significant influence on the development of spontaneous tumors in any of the organs examined in the carcinogenicity study. In conclusion, based on slight histopathological changes observed in 5% male livers, the no-observed-effect level (NOEL) was estimated to be 2.5% in the diet (1,253 mg/kg bw/day) and the no-observed-adverse-effect level (NOAEL) was determined to be 5% in the diet (2,388 mg/kg bw/day) for male rats, and for females, the NOEL was concluded to be 5% in the diet (2,826 mg/kg bw/day). Additionally, paprika color was not carcinogenic to male and female F344 rats under the present experimental conditions.

A 13-week oral dose subchronic toxicity study of gardenia yellow containing geniposide in rats.

Gardenia yellow powders A, B and C, containing geniposide at 0.284%, 0.938% and 2.783%, respectively, were administered orally to male and female SD rats as 3% feed admixtures for 13-weeks to evaluate any potential toxicity. Mean geniposide intake values were 5.72, 18.9 and 56.3mg/kg/day in groups receiving these feed admixtures, respectively. All animals survived the duration of the study. The following findings were evident in the gardenia yellow C group: chromatouria, slightly increased plasma total bilirubin, blackish brown discoloration of the kidneys and liver, brown pigments in the proximal tubular epithelium of the kidneys. Slightly increased plasma total bilirubin was considered to be due to interference of metabolite of geniposide with the system of measurement and not to be a toxic effect since there were no related changes in histopathology of the liver or in any blood chemistry parameters. Other findings were limited to pigmentations or discolorations attributable to metabolites of geniposide. No treatment-related effects were evident on body weight, food consumption, ophthalmology, hematology or organ weights in any group. Therefore, it was concluded that 3-month ingestion of the gardenia yellow powder containing geniposide at 2.783% (approximately 60 mg/kg/day as geniposide intake) does not cause any severe toxic effects.

[Research and development of Fructus Gardeniae].

Survey on research and development of Fructus Gardeniae in the recent 10 years. Gardenia yellow has been used for food colorent, medicine, feedingstuff and cosmetic. Garnedia blue has been used for developing another pigment with red and yellow. Fructus Gardeniae has been used in digestive system for cholecyst constracting and gall-stone eliminating, for declining peroxide on SAP mouse and increasing immune ability, for protecting liver against cancel, anti-acetylcholinic restraining on stomach enginery, in cardiovascular system it has been used for centrally anti-hypertension, preventing atheroma and thrombus, also Fructus Gardeniae has been used for anti-inflammation, treating parenchyma injure etc. Geniposide used for increasing production in agriculture has wider perspect.

A subchronic toxicity study of dunaliella carotene in F344 rats.

Dunaliella carotene, extracted from dunaliella alga (Dunaliella bardawil or Dunaliella salina), for use as a food-coloring agent, has beta-carotene as its mainly constituent. As there have been no reports of toxicological evaluation, a 90-day subchronic toxicity study was here performed in F344 rats at dose levels of 0 (control), 0.63%, 1.25%, 2.5% and 5% in powdered basal diet. The average daily intakes of dunaliella carotene were 352, 696, 1420 and 2750 mg/kg/day, respectively, for males, and 370, 748, 1444 and 2879 mg/kg/day for females. No mortality or treatment-related clinical signs were observed throughout the experimental period in any of the groups. Body weight gain was slightly but significantly (p < 0.05) reduced from week 5 to the end of the experiment in 2.5% and 5% males. Increased PLT were observed in 1.25% and 5% males, and 2.5% and 5% females. Significant elevations or tendencies for increase in serum T. Cho and Ca were observed in all treated males and females, with clear dose-dependence in males. Organ weight measurement and histopathological observation revealed no toxicological changes. Based on growth suppression, no-observed-adverse-effect-levels (NOAELs) were estimated to be 1.25% (696 mg/kg/day) for males and 5% (2879 mg/kg/day) for females. As increases in serum Ca were observed in the lowest group in both sexes, a no-observed-effect level (NOEL) could not be determined in this study.

Absence of carcinogenic and anticarcinogenic effects of annatto in the rat liver medium-term assay.

Annatto (Bixa orellana L.) is a natural food colorant extensively used in many processed foods, especially dairy products. The lower cost of production and the low toxicity, make annatto a very attractive and convenient pigment in substitution to the many synthetic colorants. In the present study we investigate the carcinogenic and anticarcinogenic effects of dietary annatto in Wistar rat liver using the preneoplastic glutathione S-transferase (GST-P) foci and DNA damage biomarkers. Annatto, containing 5% bixin, was administered in the diet at concentrations of 20, 200, and 1000 ppm (0.07; 0.80 and 4.23 bixin/kg body wt/day, respectively), continuously during 2 weeks before, or 8 weeks after DEN treatment (200 mg/kg body wt, i.p.), to evaluate its effect on the liver-carcinogenesis medium-term bioassay. The comet assay was used to investigate the modifying potential of annatto on DEN (20 mg/kg body wt)-induced DNA damage. The results showed that annatto was neither genotoxic nor carcinogenic at the highest concentration tested (1000 ppm). No protective effects were also observed in both GST-P foci development and comet assays. In conclusion, in such experimental conditions, annatto shows no hepatocarcinogenic effect or modifying potential against DEN-induced DNA damage and preneoplastic foci in the rat liver.

Subacute toxicity assessment of annatto in rat.

Increased human use of annatto (Bixa orellana L), a red yellow food colorant, demands generation of toxicity data. The toxic effects of annatto powder (bixin 27%) have been assessed following administration of a subacute regimen (4 weeks, 20 doses) in Wistar male and female rats. A full study with three dose levels was considered unnecessary since no sign of toxicity had been noted in a preliminary experiment with 1000 mg/kg body weight/day as was recommended by the OECD guideline. In this study, annatto administered by gavage at a dose level of 2000 mg/kg/day decreased male body weight gain, but had no effect on either food intake or food conversion efficiency. Haematological and plasma biochemical examination as well necropsy performed at the end of administration (29th day) and observation (43rd day) periods revealed no alterations related with annatto administration. Kidney apoptosis occurred in 20% treated female rats in restricted areas without proliferation or tubular segments modification. The precise nature of apoptosis was not investigated in the present study. These findings suggest that annatto was no toxic to the rat.

Resurgence of natural colourants: a holistic view.

Today, natural colourants are emerging globally, leaving synthetic colourants behind in the race, due to the realisation that are safer and ecofriendly in nature. In this context, a brief review of natural colourant sources, their classification, chemical constituents responsible for producing different colours, its activities and effect of different mordants on the hue is discussed.

A subchronic toxicity study of shea nut color in Wistar rats.

Shea nut color, obtained from nuts of the shea tree (Butyrospermum parkii), is used as a food-coloring agent. Flavonoid pigments are considered to be the responsible constituents. As there have been no reports of toxicological evaluation, a 13-week subchronic toxicity study was performed in Wistar Hannover rats at dose levels of 0 (control), 0.07, 0.31, 1.25 and 5% in powdered basal diet. The average of daily shea nut color intake was 51.3, 226.1, 986.8 and 3775.5 mg/kg/day for males and 56.4, 272.9, 1166.7 and 4387.7 mg/kg/day for females, respectively. During the administration period, daily observation of clinical signs and weekly measurement of body weights and food consumption were performed. After the end of the treatment, hematology, serum biochemistry, organ weight and histopathological examinations were conducted. No significant toxicological changes were observed in any parameters in this study. Hence, the no adverse effect dose of shea nut color was estimated to be greater than 5.0% for both sexes (3775.5 mg/kg/day for males and 4387.7 mg/kg/day for females).

Study on the mutagenicity and antimutagenicity of a natural food colour (annatto) in mouse bone marrow cells.

Most manufactured foods contain chemicals added as a deliberate part of the manufacturing process. The aims of the present study were to evaluate the mutagenicity and antimutagenicity of annatto, a natural pigment extracted from the Bixa orellana L. and widely used as a colorant in foods. The micronucleus test was performed in bone marrow cells from Swiss male mice treated with one of the three concentrations of annatto (1330, 5330 and 10,670 ppm), incorporated into the diet. The animals were fed with the diets for 7 days and sacrificed 24 h after the last treatment. For the evaluation of the antimutagenic potential of annatto, at day 7, the animals received an intraperitoneal injection of cyclophosphamide (50 mg/kg body weight). Under the concentrations tested annatto did not present mutagenic or antimutagenic activities on the mice bone marrow cells. However, an increased frequency of micronucleated cells was observed when the highest concentration (10,670 ppm) was administered simultaneously with cyclophosphamide. In conclusion, the data indicate that annatto colour, for the conditions used, is neither mutagenic nor an inhibitor of induced mutations, although it should be used carefully since high doses may increase the effect of a mutagen.

[Kooroo color: 90-day dietary toxicity study in F344 rats].

A subchronic toxicity study on kooroo color was conducted using F344 rats of both genders. Kooroo color is an extract of yam root, Dioscorea matudai Hayata, of which the major components are known to be flavonoid pigments. Use of kooroo as a food color is permitted by the Food Sanitation Law in Japan, but the chronic toxicity has not been evaluated in the literature. Rats were fed the product of kooroo color (PKC) at doses of 0.5%, 1.50%, and 5.0% in basal powder diet, while control groups received PKC-free basal diet, for ninety days. A vehicle control given propylene glycol (PG) alone, at the same dosage that the 5.0% group received, was included, because PKC used in this study contained ca. 80 percent PG, used as an extractant during the manufacturing processes. Daily observation of general behavior, and weekly measurement of body weight as well as food consumption were performed. Hematological, serum biochemical and anatomopathological examinations were conducted at the end of administration. No abnormalities ascribable to the treatment with PKC or PG were noted in any examination in this study. Hence, dietary intake of 5.0% of PKC, i.e., 2,993 mg/kg/day for males, and 3,376 mg/kg/day for females, as a mean daily intake for 90 days, had no observable adverse effect in F344 rats. Therefore, kooroo color has no significant general toxicity, and its toxicity, if any, is of a very low order.

Evaluation of the developmental toxicity of annatto in the rat.

Annatto, a dye extracted from Bixa orellana seeds, is used as a color additive in butter, cheese and in a variety of other foods as well as in drugs and cosmetics. Toxicological data on annatto and on its main carotenoid pigment bixin are still scarce. In this study we evaluated the developmental toxicity of annatto (28% of bixin). Annatto (0, 31.2, 62.5, 125, 250 and 500 mg/kg body weight/day) was given by gavage to Wistar rats on days 6-15 of pregnancy. Ceasarean sections were performed on day 21. Implantations, living and dead fetuses and resorptions were recorded. Fetuses were weighed and examined for externally-visible anomalies. One-third of fetuses from each litter was examined for visceral anomalies by a microsectioning technique. The remaining fetuses were cleared and stained with Alizarin Red S for skeleton evaluation. No adverse effect of annatto on the mothers was noted. No increase in embryolethality and no reduction of fetal body weight were observed among annatto-exposed rats. Annatto did not induce any increase in the incidence of externally-visible, visceral or skeletal anomalies in the exposed offspring. These findings suggest that annatto was neither maternally toxic nor embryotoxic in the rat. Therefore, the no-observed-adverse-effect level (NOAEL) for annatto-induced maternal and developmental toxicity was 500 mg/kg body weight/day or greater (or > or = 140 mg bixin/kg body weight/day) by the oral route.

Genotoxicity of gardenia yellow and its components.

Gardenia fruit (Gardenia jasminoides ELLIS) is widely used as a natural food colorant and as a traditional Chinese medicine for treatment of hepatic and inflammatory diseases. "Gardenia yellow" is a natural food colorant which is extracted by ethanol from gardenia fruit. The purpose of this study was to evaluate the genotoxicity of gardenia yellow. Genotoxicity of gardenia yellow and its components, crocetin, gentiobiose (a component of crocin), geniposide and genipin (formed by hydrolysis of geniposide), was studied by Ames test, rec-assay, and sister chromatid exchange (SCE) using V79 cells. Gardenia yellow and its components were found not to be mutagenic in the Salmonella reverse mutation assay. Gardenia yellow and genipin caused damage of DNA in rec-assay. Gardenia yellow induced a significant dose-dependent increase of SCE frequency (8.6 times at 1000 microg/ml as the value for the solvent control). Only genipin induced SCEs significantly among the components of gardenia yellow. Moreover, genipin induced a significant increase of tetraploids at all doses tested (95% at 8 microg/ml). Gardenia yellow preparation was analyzed by capillary electrophoresis (CE), and geniposide was detected. However, genipin was not observed. In conclusion, we have shown that genipin possesses genotoxicity. Furthermore, there were unidentified genotoxicants in gardenia yellow.

[A 90-day repeated dose toxicity study of madder color in F344 rats: a preliminary study for chronic toxicity and carcinogenicity studies].

A 90-day toxicity study of madder color was performed in F344 rats by feeding the pellet diet containing 0, 0.6, 1.2, 2.5 and 5.0% of test substance to clarify its toxic potential and to determine the dose levels for the following chronic toxicity/carcinogenicity studies. Body weight gain and food consumption were dose-dependently decreased at 1.2% or more in males and at 2.5% or more in females throughout the experimental period. All animals were survived until the end of experiment and subjected to autopsy. Hematologically, the following parameters were fluctuated in relation to the treatment: decreases in the red blood cells, hemoglobin, and hematocrit in females at 2.5% or more; increase of platelets in males at 2.5% or more, and in females at 5%; increase in white blood cells in males at 5%. Serum protein parameters were also affected by the treatment in males at 1.2% or more and in females at all doses. Increase in the serum calcium level was observed in males at 2.5% or more and in females at 5%. Serum inorganic phosphorus level was also increased in males at 1.2% or more and in females at 2.5% or more. At autopsy, both absolute and relative kidney weights of females increased dose-dependently at 0.6% or more. Relative liver weight in females also increased at 1.2% or more. Histopathologically, microvesicular vacuolar degeneration of proximal tubules was observed in the kidney of both sexes (males at 1.2% or more; females at 0.6% or more). In addition, mononuclear cell infiltration (both sexes) and hyaline casts and tubular regeneration (male) appeared in the kidney at 5%. In the female liver, focal liver cell necrosis associated with mononuclear cell infiltration was evident at 5%. The results demonstrate the toxic effects of madder color on the liver (in females at 5%) and kidney (in males at 1.2% or more; in females at 0.6% or more) of F344 rats when treated orally for 90 days. In addition, toxicities in hematopoietic system and/or bone would probably be appeared when rats are treated with 1.2% or more of madder color for long-term over 90 days. NOAEL was determined to be 0.6% in males, but could not be determined in females under the condition of this study. Based on the results of this study, the dose levels for subsequent chronic toxicity and carcinogenicity studies were determined to be 0.2, 1.0 and 5.0%, and 2.5 and 5.0%, respectively.

Subchronic toxicity study in mice fed Spirulina maxima.

The purpose of this study was to evaluate the toxicity of Spirulina maxima, a blue-green alga used as food supplement and food coloring, after 13 weeks of treatment. Groups of ten mice of each sex were given S. maxima in the diet at concentrations of 0 (control), 10, 20 or 30% (w/w) for 13 weeks. The alga ingestion had no effect on behavior, food and water intake, growth or survival. Terminal values in hematology and clinical chemistry did not reveal differences between treated and control groups. However, male and female mice showed significant changes in serum cholesterol levels at 20 and 30% algal concentrations, but a toxic effect of S. maxima was excluded. Post-mortem examination revealed no differences in gross or microscopic findings. Our results show that S. maxima up to high feeding levels did not produce adverse effects in mice after subchronic treatment.